AAC Accepted Manuscript Posted Online 27 July 2015 Antimicrob. Agents Chemother. doi:10.1128/AAC.00899-15 Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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The Glucose Transporter PfHT is an Antimalarial Target of the HIV Protease Inhibitor Lopinavir

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Running title: HIV Drug Lopinavir Blocks Malarial Glucose Transport

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Thomas E. Krafta, Christopher Armstronga, Monique R. Heitmeiera, Audrey R. Odoma,c and Paul

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W. Hruza,b#

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a

Department of Pediatrics, and bDepartment of Cell Biology and Physiology, cDepartment of

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Molecular Microbiology, Washington University School of Medicine, St Louis MO 63110

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# Address all correspondence to Paul W. Hruz.: [email protected]

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Abstract

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Malaria and HIV infection are co-endemic in a large portion of the world and remain a major

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cause of morbidity and mortality. Growing resistance of Plasmodium species to existing

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therapies has increased the need for new therapeutic approaches. The Plasmodium glucose

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transporter PfHT is known to be essential for parasite growth and survival. We have previously

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shown that HIV protease inhibitors (PIs) act as antagonists of mammalian glucose transporters.

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While the PI lopinavir is known to have antimalarial activity, the mechanism-of-action is

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unknown. We report here that lopinavir blocks glucose uptake into isolated malaria parasites

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at therapeutically relevant drug levels. Malaria parasites depend on a constant supply of

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glucose as their primary source of energy and decreasing the available concentration of glucose

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leads to parasite death. We identified the malarial glucose transporter PfHT as a target for

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inhibition by lopinavir that leads to parasite death. This discovery provides a mechanistic basis

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for the antimalarial effect of lopinavir and provides a direct target for novel drug design with

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utility beyond the HIV-infected population.

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Introduction Despite aggressive world-wide efforts to eradicate malaria, this life-threatening disease

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continues to affect over 200 million people per year, resulting in an annual death toll exceeding

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half a million, mostly among African children(1). Currently, vaccination against malaria is not

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available, while resistance against all known therapeutics is spreading(1). As a result, newer

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antimalarial agents with novel mechanisms-of-action are urgently needed.

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The global prevalence of malaria and HIV infection largely overlap geographically.

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Combination antiviral therapy that includes the HIV protease inhibitor (PI) lopinavir has been

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found to dramatically decrease malaria incidence in a pediatric clinical population by 41%,

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suggesting a direct effect of PIs on parasite replication(2). Indeed, lopinavir has demonstrated

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in vitro activity(3) against Plasmodium falciparum, the protozoan species responsible for most

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malaria deaths(1, 4). In addition, lopinavir reduces the malaria liver stage burden in infected

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rhesus monkeys in vivo at clinically relevant concentrations(5). Despite ongoing efforts, the direct cellular target(s) of lopinavir responsible for its

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antimalarial properties against P. falciparum remains unclear. PIs were originally designed as

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antagonists of the viral aspartyl protease(6). The malaria parasite requires a class of aspartyl

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proteases called plasmepsins, which are necessary to degrade host hemoglobin(7) and direct

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export of malaria export proteins(8); however the antimalarial activity of PIs does not appear to

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be mediated through plasmepsin inhibition(9, 10). Identifying the antimalarial mechanism-of-

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action of PIs is imperative for finding a novel, clinically proven drug target and developing a new

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class of lopinavir-like antimalarial drugs. In clinical populations, prolonged use of PIs is associated with insulin resistance. Recent

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studies have identified the molecular mechanism of this effect, which is mediated by direct

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binding of PIs to the insulin-responsive facilitative glucose transporter GLUT4(11–13). The

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human glucose transporters share sequence homology with the essential P. falciparum glucose

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transporter, PfHT. Similar to GLUT1 and GLUT4, the predicted topology of PfHT comprises 12

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transmembrane helices, forming a central glucose permeation path. Key residues that are

3

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involved in glucose binding and transport are preserved between the human and malaria

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glucose transporters(14, 15). Intraerythrocytic malaria parasites depend on a constant supply of glucose as their

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primary source of energy(16). Not surprisingly, infected erythrocytes show a ~100 fold increase

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in glucose consumption compared to uninfected erythrocytes(17). PfHT (PF3D7_0204700) is the

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principal glucose transporter, transcribed from a single copy gene with no close paralogue(14).

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PfHT has been genetically validated as essential in Plasmodium parasites(18) and has been

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independently chemically validated as a novel drug target against malaria(14, 19). Here we show that lopinavir inhibits glucose uptake into the P. falciparum parasite by

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blocking PfHT at therapeutically relevant concentrations. This establishes a direct molecular

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target for the antimalarial activity of lopinavir and validates the utility of targeting PfHT in novel

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drug development.

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Material and Methods

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Materials

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[14C]-2-deoxy- glucose (2-DOG) was purchased from PerkinElmer. [3H]-2DOG was purchased

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from American Radiolabels Inc. PfHT DNA was codon optimized and synthesized by Life

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Technologies (Grand Island, NY). GLUT1 shRNA was obtained through the RNAi core at

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Washington University, School of Medicine. HEK293 cells were acquired from the American

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Type Culture Collection. HIV protease inhibitors were obtained through the NIH AIDS Reagent

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Program, Division of AIDS, NIAID, NIH. Compound 3361 was kindly donated by Sanjeev Krishna

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(Centre for Infection, Division of Cellular and Molecular Medicine, St. George's, University of

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London).

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Malaria tissue culture

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The P. falciparum strain strain 3D7 was obtained from the Malaria Research and Reference

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Reagent Resource Center (MR4, ATCC, Manassas, Virginia). Unless otherwise stated, P.

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falciparum strains were cultured at 37°C in a 2% suspension of human erythrocytes in RPMI-

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1640 medium (Sigma-Aldrich, SKU R4130) supplemented with 27 mM sodium bicarbonate, 11

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mM glucose, 5 mM HEPES, 1 mM sodium pyruvate, 0.37 mM hypoxanthine, 0.01 mM

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thymidine, 10 µg/mL gentamicin, and 0.5% Albumax (Life Technologies) in a 5% O2/ 5% CO2 /

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90% N2 atmosphere, as previously described(20, 21). Culture growth was monitored by

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microscopic analysis of Giemsa-stained blood smears.

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Drug and glucose sensitivity of P. falciparum

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Asynchronous P. falciparum cultures were diluted to 1% parasitemia and were treated at a

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range of concentrations of inhibitor or glucose. Growth inhibition assays were performed in

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opaque 96-well plates at 100 µL culture volume. After 3 days, parasite growth was quantified

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by measuring DNA content using Picogreen (Life Technologies), as previously described(22).

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Picogreen fluorescence was measured on a FLUOstar Omega microplate reader (BMG Labtech)

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at 485 nm excitation and 528 nm emission. IC50 values were calculated by nonlinear regression

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analysis using GraphPad Prism software.

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Stable expression of PfHT

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HEK293 cells were stably transfected with codon optimized PfHT DNA in the pcDNA 3.1(-) hygro

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plasmid (Life Technologies) as described earlier(23). After selection with hygromycin, 10

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colonies were grown to near confluency in 4cm tissue culture dishes and the highest expresser

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of PfHT was selected using [3H]-2DOG uptake and quantitative RT-PCR (as described below).

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Short hairpin RNA lentiviruses for GLUT1 knockdown

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293T packaging cells were cotransfected with VSVg and Delta 8.9 packaging plasmids plus

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shRNA lentiviral plasmids targeting GLUT1 (NM_006516.1-2310s1c1) using Optifect transfection

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reagent (Life Technologies). The media was changed 12 hours after transfection and

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supernatants (10 ml) were harvested every 24 hours for 72 hours and kept at 4°C until they

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were pooled, filtered through 0.45 μm syringe filters, aliquoted and stored at -80°C until use.

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HEK293 wild-type cells or HEK293 cells stably expressing PfHT were infected by exposing them

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to viral supernatant for 48 hours, then replacing with selection media (containing 5 μg/ml

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puromycin).

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Quantitative PCR analysis

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Total RNA was isolated using the TRIzol Plus RNA Purification System (Life Technologies). RNA

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was reverse transcribed using the qScript cDNA SuperMix (Quanta Biosciences, Gaithersburg,

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MD). Quantitative RT-PCR was performed using the Power SYBR® Green PCR Master Mix

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(Applied Biosystems) as described previously(24).

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2-DOG uptake into P. falciparum and HEK293 cells

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Uptake of [14C]-2DOG into isolated parasites was determined at room temperature using the

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methods described previously(25). Lopinavir and compound 3361 were added 5min prior to the

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addition of [14C]-2DOG (0.2 µCi/mL final concentration). Uptake of [3H]-2DOG into HEK293 cells

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was measured in phosphate buffer at 37°C for 5min as described previously (26).

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Statistical analyses

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The data are reported as means ±SEM. Differences between control and experimental values

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were determined by one-way ANOVA analysis.

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Modeling of PfHT and docking of lopinavir

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PfHT structure was predicted using the I-TASSER webserver(27–29). The webserver was

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amongst top scorers of the Server Section of the 7th (2006), 8th (2008), 9th (2010), 10th CASPs

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(2012), and 11th CASPs (2014), a community-wide experiment for testing the state-of-the-art of

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protein structure predictions. We chose a model that resembled the inward open

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conformation. Docking was performed using AutoDock Vina(30). The receptor was prepared

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using MGLTools version 1.5.6 and the ligand lopinavir was prepared using ArgusLab. The

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docking process was repeated with number of modes of 50 and increasing exhaustiveness of

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25, 50 and 100. The results in each attempt were 20 similar docked poses. Docked poses were

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displayed using PyMOL version 1.7.4.0.

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Results

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We compared the effect of currently FDA-approved HIV protease inhibitors on the

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replication and survival of P. falciparum parasites in asexual erythrocyte cell culture. Consistent

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with prior reports(3, 9) , all tested compounds inhibited parasite growth. Lopinavir was the

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most potent inhibitor with an IC50 of 1.9 μM (Fig. 1a). We previously determined the inhibitory

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effect of several HIV protease inhibitors on human glucose transport(11, 13, 26). Since HIV PIs

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inhibit human glucose transport, we evaluated whether lopinavir might inhibit glucose uptake

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in isolated P. falciparum parasites. We compared the uptake of radiolabeled 2-DOG in live

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parasites, isolated from erythrocytes treated with vehicle, lopinavir, or the chemically unrelated

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PfHT inhibitor 3361 (Fig. 2a)(19). The O-3-hexose derivative compound 3361 [3-O-((undec-10-

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en)-yl)-D-glucose)] has previously been shown to selectively inhibit PfHT relative to several

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mammalian sugar transporters and inhibit glucose uptake by the parasite, leading to inhibition

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of growth and proliferation in vitro and in vivo(19, 25). We found that high concentrations of

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lopinavir potently inhibited uptake of radiolabeled glucose by more than 70% compared to

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vehicle-treated control, similar to the effect observed by the known PfHT inhibitor compound

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3361. In dose-response studies (Fig. 2b), we found that the half-maximal inhibitory

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concentration (IC50) of lopinavir was 16 ± 4.2 μM, which is within the range of average serum

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peak levels of both adult and pediatric patients treated with lopinavir/ritonavir(31–34). To confirm that inhibition of malaria glucose transport was mediated through PfHT

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inhibition, we developed and tested the effect of this PI on glucose transport in a PfHT-

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overexpressing HEK293 cell line. HEK293 cells predominantly transport glucose using the

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facilitative transport protein GLUT1. Using a PfHT sequence codon-optimized for mammalian

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cell expression, we overexpressed the malarial hexose transporter protein and knocked down

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the expression of GLUT1 using shRNA. In this engineered cell line, PfHT was the predominant

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hexose transporter expressed (Fig. 3a). Glucose uptake in the PfHT overexpressing cell line was

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>5 times higher compared to background cells that did not overexpress PfHT but were

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transduced with GLUT1 shRNA (Fig. 3b). We quantified the amount of [3H]-2-DOG accumulated

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in PfHT overexpressing HEK293 cells in the presence of varying concentration of lopinavir and

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determined an IC50 of 14 ± 2 μM. The IC50 values for the effect of lopinavir on glucose uptake in

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both P. falciparum parasites and PfHT-overexpressing HEK293 cells are closely correlated,

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consistent with a model in which inhibition of PfHT is responsible for decreased glucose uptake

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and reduced parasite growth (Fig. 4). Supporting this model, blood stage P. falciparum is

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acutely sensitive to reduced extracellular glucose concentrations (17). Since the measurement

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of 2-DOG uptake involves both the transport of this sugar and phosphorylation to 2-DOG-6-P,

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we cannot fully exclude the possibility that lopinavir also inhibits the malarial hexokinase.

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However, the observation that lopinavir inhibits 2-DOG uptake with different IC50s when

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measured in HEK293 cells overexpressing different GLUTs (ranging from 3μM for GLUT4 to

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32μM for GLUT1) suggests that hexokinase is not directly targeted. To further investigate the mode of action of lopinavir on PfHT, we determined several

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parameters by Michaelis-Menten kinetics. As expected, uptake of glucose is saturated with

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increasing substrate concentration. The observed increase in Km with constant Vmax indicates

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that lopinavir acts as a competitive inhibition of glucose uptake (Fig. 5). To investigate the binding site of lopinavir on PfHT, we generated a model of the

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transporter based on several crystal structures of homologous proteins using the I-TASSER

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webserver (Table 1). We selected a model with high cluster density in an inward conformation

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to determine the lopinavir binding site by docking analysis. We created a docking volume

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covering the entire protein to obtain unbiased results. Lopinavir was docked to the PfHT model

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using AutoDock VINA via rigid receptor, flexible ligand docking. All docked poses of lopinavir

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were bound to single binding pocket on the intracellular side of the glucose permeation path,

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preventing glucose from entering and reaching its binding site (Fig. 6a). These results could be

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replicated in several docking experiments using random seeds and different degrees of

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exhaustiveness, strongly supporting this position as the lowest energy binding site. We

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compared the PfHT-lopinavir putative binding site with the binding site of the structurally

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related protease inhibitor indinavir in the PfHT homologue GLUT4, published by Hresko et al.

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(Fig. 6b)(26). The docking experiments suggest that both indinavir and lopinavir bind to a

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structurally similar binding pocket in their respective glucose transporters. Lopinavir binding

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within the glucose permeation pathway is consistent with this drug acting as a competitive

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inhibitor of zero-trans glucose uptake (Fig. 5). Although inhibition of GLUT4 by Indinavir

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requires drug binding from the intracellular side of the transporter (35), lopinavir appears to

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have access to this domain from the exofacial side of PfHT. Similar to the observed differences

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in isoform-selectivity of HIV protease inhibitors for mammalian GLUTs (26), it is possible that

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steric influences of amino acid side chains lining the glucose permeation pathway contribute to

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the ability of these drugs to target PfHT.

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Discussion

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There is a compelling need for novel therapies for treatment of malaria. In particular,

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the emergence and spread of resistance to artemisinin-based compounds threatens malaria

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control efforts worldwide. Protease inhibitors, such as lopinavir, represent promising

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therapeutic leads for new antimalarial development. Lopinavir has potent antimalarial activity

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in vitro and in animal models(4, 5), and human studies of lopinavir-treated individuals in

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endemic areas indicate antimalarial potency at clinically relevant doses(2). Importantly, many

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of the pharmacologic challenges that slow antimalarial development have already been

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overcome with lopinavir, which is orally bioavailable, available in suspension form (for pediatric

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use), and suitable for once-daily dosing in combination with ritonavir. Our studies provide substantial evidence that inhibition of the malaria hexose

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transporter, PfHT, is responsible for the antiparasitic effects of lopinavir. PfHT hexose

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transporter function is known to be required for Plasmodium parasite development in culture

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and in animal models(14, 18, 19, 25, 36). We find that not only does lopinavir directly block

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glucose uptake into P. falciparum parasites, it also inhibits glucose uptake in human cells

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engineered such that majority of hexose transport is through heterologously expressed PfHT.

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Importantly, profound reductions of glucose uptake into parasites and into PfHT-expressing

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human cells are achieved at lopinavir concentrations well below therapeutically achieved drug

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levels(31–34).

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Of note, even lower concentrations of both lopinavir and compound 3361 are required

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to inhibit parasite replication than are required to inhibit glucose transport(19). This difference

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may be explained by the reliance of parasites on a constant glucose supply. Plasmodium

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infection increases cellular glucose consumption in erythrocytes nearly 100 fold, while parasite

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survival is highly sensitive to reduced extracellular glucose concentrations(17). Thus, even

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partial inhibition of Plasmodium glucose transporters may lead to growth inhibition and

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eventually death of the parasite. PfHT may therefore represent a promising, parasite-specific

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“Achilles Heel” for target-based drug discovery efforts. We cannot rule out the possibility that

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there are additional targets of lopinavir in P. falciparum other than PfHT . The existence of

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additional drug targets, if present, could delay the development of drug resistance, a major

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problem for malaria drugs. However, observing this difference for both lopinavir and

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compound 3361, two structurally unrelated drugs that inhibit PfHT-mediated glucose transport

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decreases the likelihood that they would both also inhibit another secondary target. As agents designed for long-term suppression of HIV replication, lopinavir and other

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protease inhibitors are well tolerated, with minor adverse events (mostly gastrointestinal)

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associated with short-term use(37). During chronic HIV treatment regimens that include PIs,

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disturbances in glucose homeostasis and other metabolic changes that increase cardiovascular

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risk have been reported(38). In this HIV-infected population, it has been challenging to

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dissociate the direct contributions of PIs from the influences of viral infection, other drug

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exposures (e.g. nucleoside reverse transcriptase inhibitors), and associated environmental

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factors. Nevertheless, the in vitro effects of PIs on the insulin-responsive facilitative glucose

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transporter GLUT4 correlate directly with in vivo changes in insulin sensitivity, both in rodent

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models(39) and in humans(40). Fortunately, the effect of PIs on glucose tolerance is readily

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reversible following short-term drug exposure, as required for treatment of malaria(39, 40).

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However, the full spectrum of PI-mediated effects on the remaining GLUT isoforms remains

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unknown, and not all PIs appear to have the same effect on GLUT4 activity(41). Ideally, the

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development of drugs that selectively target PfHT over mammalian GLUTs would provide highly

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selective inhibition of PfHT over the mammalian GLUTs, resulting in a superior safety profile. The development of high-throughput screening protocols targeting the Plasmodium

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glucose transporter will help identify additional classes of PfHT antagonists. Several assays have

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been developed by expressing PfHT in yeast or Leishmania(14, 18, 42). In combination with

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these prior studies, our current findings confirm that PfHT is an attractive, druggable target for

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malaria. Our development of a PfHT overexpressing HEK293 cell line and demonstration that

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this system can reliably test for drug-mediated inhibition of the malarial glucose transporter

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can significantly aid candidate drug screening. Complementing a high-throughput approach is

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the opportunity for structure-based design. This can allow for higher affinity and selectivity to

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the plasmodium transporter while minimizing effects on mammalian glucose transporters

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(GLUTs). GLUT1 is the most extensively characterized of the human GLUTs and a crystal

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structure of this protein was recently reported(43). In the absence of structural data for PfHT

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and the other known GLUTs, it has been necessary to rely upon molecular modeling based on

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the structures of related bacterial and mammalian transporter. Although, our docking results

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are consistent with the previously identified HIV protease inhibitor binding site in the closely

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related transporter GLUT4(26), we cannot exclude the possibility that lopinavir binds to a

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distinct binding site only accessible in an outward open conformation of PfHT. Testing this

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possibility will generate valuable data once a homologues transporter is crystallized in the

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outward open conformation. Future efforts to establish a comprehensive structure-activity

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profile for the inhibition of PfHT and the human GLUTs may provide valuable information about

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the feasibility of this approach. Investigation of potential additive or synergistic effects of PfHT

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inhibitors with existing anti-malarial agents or other compounds that inhibit parasite

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metabolism will also be of great interest. In summary, the identification of lopinavir as a clinically meaningful inhibitor of the

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essential malaria hexose transporter PfHT provides a strong rationale and molecular basis for

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future antimalarial development efforts targeting this protein. Because PfHT inhibition retards

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both the liver and mosquito-stage development of rodent malaria parasites(44), novel PfHT

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inhibitors hold great promise for use in malaria treatment and as transmission-blocking agents.

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The existing pharmacokinetic and safety data for lopinavir should allow rapid assessment of the

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suitability and efficacy of this agent in non-HIV infected patient populations. Understanding the

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molecular target of lopinavir in P. falciparum will be key to any necessary medicinal chemical

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optimization of lopinavir and related protease inhibitors, repurposed for use as antimalarials.

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Acknowledgements

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This work was supported by the Children’s Discovery Institute of Washington University and St.

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Louis Children’s Hospital (MD-LI-2011-171, to A.R.O.), NIH/NIAID R01AI103280 (to A.R.O.), a

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March of Dimes Basil O’Connor Starter Scholar Research Award (to A.R.O.), and a Doris Duke

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Charitable Foundation Clinical Scientist Development award (to A.R.O.).

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The authors thank Maria Payne for conducting quantitative PCR experiments and generating

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plasmids.

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Author Contributions

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T.E.K. and P.W.H. conceived the initial idea of the study. T.E.K., C.A. and M.H. performed the

422

experiments. T.E.K. created the model and performed the docking. T.E.K., A.R.O. and P.W.H.

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contributed to the experimental design and data analysis. T.E.K., A.R.O. and P.W.H. wrote the

424

manuscript with feedback from M.H.

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Competing Financial Interests statement

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The authors declare no competing financial interests.

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Figure Legends

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Figure 1: Comparison of HIV protease inhibitors’ potency on P. falciparum growth inhibition (a)

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IC50 comparison of HIV protease inhibitors on growth P. falciparum in blood culture. IC50 was

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calculated using non-linear regression analysis and is expressed as means with 95% confidence

435

interval. (b) Chemical structures of the three most potent inhibitors HIV protease inhibitors of

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PfHT mediated glucose uptake.

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Figure 2: (a) Uptake of [14C]2DOG by isolated P. falciparum trophozoites in the presence of

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lopinavir (100 μM), compound 3361 (200 μM) or vehicle. The parasites were suspended in

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HEPES-buffered saline containing 200 μM glucose. (b) Uptake of [14C]2DOG by isolated P.

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falciparum trophozoites at increasing concentrations of lopinavir. The uptake was quenched

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after 2 minutes. IC50 was calculated using non-linear regression analysis. Uptake data are

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expressed as means ±SEM. A 30sec or 1min time point would have been a more accurate

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measurement of initial rates however, we chose the longer time point to enhance the uptake

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signal as the degree of non-linearity will probably not greatly affect the calculated IC50 value.

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Figure 3. A. mRNA expression levels of glucose transporter proteins in HEK293 wild-type (WT),

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GLUT1 knock-down (KD) and GLUT1 KD + PfHT overexpressing (OE) cells. mRNA levels were

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determined via quantitative PCR and levels are displayed in copy number per ng of cDNA. Data

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are expressed as means ±SEM. B. Comparing specific activity of glucose uptake in HEK293 cells

21

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expressing GLUT1 shRNA and plus and minus overexpression of PfHT. Data are expressed as

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means ±SEM (*P